Kartogenin (KGN)/synthetic melanin nanoparticles (SMNP) loaded theranostic hydrogel scaffold system for multiparametric magnetic resonance imaging guided cartilage regeneration

Abstract Cartilage regeneration after injury is still a great challenge in clinics, which suffers from its avascularity and poor proliferative ability. Herein we designed a novel biocompatible cellulose nanocrystal/GelMA (gelatin‐methacrylate anhydride)/HAMA (hyaluronic acid‐methacrylate anhydride)‐blended hydrogel scaffold system, loaded with synthetic melanin nanoparticles (SMNP) and a bioactive drug kartogenin (KGN) for theranostic purpose. We found that the SMNP‐KGN/Gel showed favorable mechanical property, thermal stability, and distinct magnetic resonance imaging (MRI) contrast enhancement. Meanwhile, the sustained release of KGN could recruit bone‐derived mesenchymal stem cells to proliferate and differentiate into chondrocytes, which promoted cartilage regeneration in vitro and in vivo. The hydrogel degradation and cartilage restoration were simultaneously monitored by multiparametric MRI for 12 weeks, and further confirmed by histological analysis. Together, these results validated the multifunctional hydrogel as a promising tissue engineering platform for noninvasive imaging‐guided precision therapy in cartilage regenerative medicine.

to gain self-repair after injury. 2 Traditional surgical procedures for articular cartilage injuries include debridement, bone marrow stimulation, autogenous osteochondral transplantation, allogeneic osteochondral transplantation, chondrocyte implantation, and joint replacement. 3 However, the efficacy of these methods remains unsatisfactory in the clinical treatment of minor articular cartilage symptoms. Debridement and bone marrow stimulation techniques (such as microfractures), as the first-line treatment, are prone to the formation of fibrocartilage that cannot withstand the articular stress over time. 4 Other widely used techniques like osteochondral grafts or chondrocyte implantation are limited by donor site morbidity, inadequate donor supply, graft failure, and stratification. 5 Recently, tissue engineering strategies containing combinations of stem cells, biomaterials (scaffolds) and/or functional biomolecules have raised interest in the field of articular cartilage repairment. 6 Bioactive factors are widely used to promote the differentiation of bone marrow mesenchymal stem cells (BMSCs) to chondrocytes. 7 Particularly, kartogenin (KGN) is a small non-protein compound that induces mesenchymal stem cells to homing. 3 Some attempts have been conducted to promote chondrocyte differentiation effectively by KGN in the treatment of osteoarthritis. 5,8,9 However, injecting KGN into the articular cavity directly will face problems like loss of KGN or absorption into the circulatory system. 10 Consequently, a continuous-release system is needed to sustainably prolong the activity of KGN to repair the damaged cartilage. More recently, Xu et al. 11 prepared biocompatible and degradable scaffold loaded with KGN and BMSCs, which facilitated a permanent release of small growth factors and enhanced chondrogenesis of encapsulated BMSCs. In addition, supramolecular injectable hydrogel containing stem cells can effectively promote hyaline cartilage and subchondral bone regeneration in a rat model of cartilage defect. 12 As is known to all, it is important to find noninvasive methods to monitor the functionalization and degradation of tissue engineering scaffolds longitudinally in vivo. Onofrillo et al 13  Magnetic resonance imaging (MRI) has been widely used to assess the morphological changes of biomaterial degradation and new tissue reconstruction for its safety and noninvasive measurement. 15 It has obvious advantages in the evaluation of various regeneration strategies with ideal soft-tissue resolution and penetration depth. 16 For example, Hong et al 17 18 Compared with traditional iron oxide nanoparticles, synthetic melanin nanoparticles (SMNP) have excellent biocompatibility and ability to coordinate separation of paramagnetic metal centers. 19 The functional catechol network of SMNP facilitates the scaffold for paramagnetic metal ion chelation to be applied to a T 1 weighted MRI contrast agent. 20 Here, we synthesized an injectable GelMA/HAMA/CNC hydrogel scaffold incorporated with KGN and SMNP ( Figure 1). The biofunctional hydrogel scaffold system can provide a microenvironment matrix to support sustained release of KGN, which promotes the homing of BMSCs and induces chondrogenic differentiation. Moreover, the cartilage regeneration and SMNP-KGN/Gel degradation were evaluated by quantitative MRI analysis longitudinally in a rabbit articular cartilage defect model. The in vivo imaging results were further confirmed by histological and pathological analysis.

| Characterization of the hydrogel
The FTIR spectrum (Figure 2a) showed the successful synthesis of both GelMA and HAMA. FTIR spectra showed an absorption peak of twisting vibration attributable to a CH 2 group at 1029 cm À1 , and absorption peak at 1610 cm À1 attributed to stretching vibration of C=C group, indicating a presence of methacryl groups in the chemical structure of GelMA. 21 Besides, the absorption peak at 1031 and 1606 cm À1 also appeared in the spectra of HAMA. Furthermore, 1 H NMR of the GelMA ( Figure 2b) and HAMA (Figure 2c) exhibited the methacrylamide vinyl group signal that increased at 6.0 ppm and 5.0 ppm, indicating MA modified the gelatin and hyaluronic acid successfully. 22 From Figure S1, the pore size distribution of the Gel-0-CNC, Gel-1-CNC and Gel-2-CNC hydrogel was 327 ± 80.37 μm, 234 ± 60.66 μm and 174 ± 60.25 μm, respectively. The prepared hydrogel was suitable for cell metabolism and cartilage regeneration. 23 The effect of different composition on the rheological properties of the hydrogel was explored by the storage modulus (G 0 ) and loss modulus (G 00 ). All hydrogels could maintain a gel state with the time From this, the Gel-1-CNC hydrogel was chosen to explore the subsequent studies. Then, the Gel-1-CNC was subjected to 100 cycles of loading/unloading by applying a 50% strain (Figure 2g).
The hydrogels produced a stress-strain curve like the original hydrogels, proving that they exhibited good anti-fatigue performance.
Moreover, suitable degradation rate of hydrogels is also a significant consideration for biomaterials. 24 In this study, we explored the degradation rates of Gel-1-CNC hydrogels in PBS and collagenase solution ( Figure 2h and Figure S2). The mass degradation rate of Gel-1-CNC was 47.8% in the in PBS and 53.6% in the hyaluronidase solution after 28 days at pH = 7.4. The Gel-1-CNC hydrogels showed higher degradation rate in collagenase, mainly because the main chain of gelatin is destroyed. 25

| Characterization of SMNP and SMNP-KGN nanoparticle
The SMNP with natural function and structure maintain lots of ideal properties of natural melanin. SMNP have tremendous potential in its utility across an extensive range of biomedical applications. 26 The SMNP and SMNP-KGN surface morphologies were observed by TEM.
The SMNP had a diameter of 188 ± 19.76 nm (Figure 3a Figure 3d, the characteristic peak at 1670 cm À1 that presented in SMNP-KGN was by reason of the F I G U R E 1 Schematic illustration of preparation and functionalization of kartogenin loaded and SMNP labeled multifunctional hydrogel scaffold for cartilage regeneration hydrazone bonds formed between SMNP and KGN via amide reaction. 27 The characteristic peak at 647 cm À1 , which was attributed to  28 According to the standard curve Figure S4, the KGN released from the SMNP-KGN/Gel reached 41% after 7 days. As shown in Figure S5, the SMNP-KGN incorporated hydrogels were scanned by T 1 -weighted MR imaging in vitro. With the increase of Fe concentration, the contrast effect of hydrogels on T 1 -weighted MRI was significantly enhanced. To study the T 1 relaxation (r 1 ), the Fe concentration in SMNP-KGN was used to fit the curve (1/T 1 relative to Fe concentration). The r 1 value was calculated as 7.95 mm À1 s À1 according to the corresponding fitting line slope.

| The biocompatibility and bioactivity of SMNP-KGN/Gel in vitro
To verify the biocompatibility of the SMNP labeled hydrogels in vitro, indicating low cytotoxicity of the as-prepared hydrogel. Interestingly, on day 7, BMSCs were flat and fusiform, and aggregated into condensed clusters for chondrogenic differentiation. 29 In the cell migration experiment, BMSCs stimulated by KGN migrated faster in the wound healing model (Figure 4d), which suggested that the KGNloaded hydrogels can facilitate host BMSCs homing to enhance hydrogel-host tissue integration. 30 These cellular experiments demonstrated that the SMNP-KGN/Gel had good biocompatibility and could promote the proliferation and migration of BMSCs in vitro. 31,32 The expression levels of chondrogenic markers genes (aggrecan, Sox9, and collagen II) and a dedifferentiation-related gene (collagen I) in the hydrogels were evaluated. As shown in Figure 4e, the expression of aggrecan, Sox9, and collagen II were significantly enhanced in the SMNP-KGN/Gel after 14 days. This can be attributed to the fact that KGN activates protein transcription, such as type II collagen and aggrecan, involved in cartilage matrix components. 33 In contrast, the expression of collagen I remained in low value (relative mRNA expression = 0.69) in the SMNP-KGN/Gel, which indicated that the KGN-loaded hydrogel promotes BMSCs for chondrogenic differentiation instead of the fibrocartilage phenotype. 34 These results indicated that the SMNP-KGN hydrogel could enhance BMSCs chondrogenesis in vitro.

| MRI and macroscopic evaluation in vivo
To investigate the efficacy and degradation of SMNP-KGN/Gel in vivo, multiparametric MRI was performed. At week 3, 6, 9, and Repair Society (ICRS) macroscopic scores were used for regeneration evaluation. 36 The osteochondral regeneration was evaluated in the aspect of total score, structural characteristics, degree of integration, joint surface regularity, safranin-O staining, and subchondral morphology. As shown in Figure 5e, the score of the KGN-treated group was significantly higher than that of other groups in week 6 and 12, which correlated well with the changes of MRI signal.

| Histological evaluation in vivo
The process of biomaterial absorption and cartilage regeneration was

| DISCUSSION
With the development of cartilage tissue engineering, hydrogels containing growth factors are widely used for cartilage repair. 37 Hydrogels with three-dimensional expansion network facilitate mesenchymal stem cells migration and provide an ideal microenvironment for their proliferation and differentiation. 30 In this study, we developed an injectable GelMA/HAMA/CNC hydrogel formed by lights, which was highly biocompatible and flexible to various shapes of defects for in vivo applications.
The ideal tissue engineering scaffold should be biodegradable to facilitate the regenerated tissue completely replaced by the graft. Previous studies have proved that HA is an ideal choice for the manufacture of cartilage regeneration engineering materials, because its degradation and drug release can adapt to the biochemical process in vivo. 38 Our H&E staining results also demonstrated that GelMA/ HAMA/CNC hydrogels could be completely degraded and absorbed in animal model. Normally, the mechanical strength of hydrogel is not as high as that of normal cartilage. 30 The enhancement effect of CNC fibers on the mechanical properties of polymers has been reported previously. 39 In this study, we found that 1% of CNC addition increased the mechanical strength of hydrogels and maintained the three-dimensional network structure of water swelling polymers stable.
KGN, a small molecule drug promoting cartilage formation first reported in 2012, 8 can effectively induce hMSCs to differentiate into chondrocytes. Some studies have shown that KGN is stable at room temperature and has good biocompatibility with no significant toxicity to normal cells. 40 44 In this study, a KGN loaded hydrogel was designed to support in situ cartilage regeneration. The ICRS score was significantly different between the KGN group and non-KGN group. All of these results suggest that KGN is expected to be an ideal drug to promote cartilage repair.
Although KGN loaded hydrogels can effectively promote cartilage regeneration, the real-time and noninvasive visualization of its in-situ fate is important. Patients may require repetitive interventions if the implants degrade too fast. As we know, X-ray is a popular choice for follow-up clinically. However, it failed to track the fate and function of the implanted materials for its poor spatial resolution. 45 Nowadays, effective methods such as fluorescence imaging 13 and photoacoustic imaging 14 to monitor the grafts or cartilage status have been studied.
But they were unable to evaluate the implants and chondrogenesis simultaneously. MRI is an appropriate imaging tool to monitor the structural and biochemical changes of articular cartilage. 46 Cartilage thickness, T 2 and T 1q relaxation time have been proved to be potential imaging markers, which can provide insight into the degree of cartilage injury and dysfunction. 47

| Preparation of SMNP
The synthesis method of SMNP was followed by the reported method. 2 Briefly, dopamine hydrochloride (45 mg) and 6.2 mg of FeCl 3 Á6H 2 O were fully dissolved in 130 ml of deionized water under stirring at 25 C for 1 h. Next, 450 mg Tris (2-amino-2-hydroxymethylpropane-1, 3-diol) was dissolved in 20 ml deionized water, then quickly injected into the established solution and reacted for 2 h. Finally, the SMNP was separated by centrifugation and washed three times with deionized water.

| Preparation of SMNP-KGN
Hydrophobic KGN powder (10 mg) was dissolved in 10 ml solution (acetone: H 2 O = 1:9) with EDC/NHS (5% w/w) for carboxyl activation. Next, 10 mg SMNP was put into the above KGN solution. We obtained a homogeneous mixture by sonicating for 30 min. Next, the solution was shaken (120 r/min) at 37 C for 24 h in a shaker. The precipitated particles were collected by centrifuging (8000 r/min), and then frozen at À20 C. Finally, SMNP-KGN NPs were obtained under lyophilization (À80 C).

| Preparation of SMNP-KGN/Gel
The preparation of SMNP/Gel and SMNP-KGN/Gel hydrogels were shown as follow: 10 mg of SMNP were dissolved in 10 ml of PBS buffer and ultrasonicated for 10 min. Next, 1000 mg of GelMA, 500 mg of HAMA, and 100 mg CNC were put into the above solution and stirred for 30 min. After 0.1% (w/v) photo-initiator LAP was added, the SMNP/Gel hydrogel was formed by irradiated 30 s under 405 nm-blue light. The SMNP-KGN/Gel hydrogel was obtained following the same procedures.

| In vitro degradation behavior
The Gel-1-CNC hydrogels were placed in PBS (pH = 5.5 or 7.4) with or without 0.02 U/mL collagenase to evaluate the degradation behavior at 37 C, respectively. The degradation ratio was calculated according to the following formula: In this equation, the W D represents the initial weight of the hydrogels, and W d is the weight after degradation at predetermined time points.

| Histological evaluation of repaired cartilage
The cartilage repair was evaluated by gross images of the femur condyles collected at week 6 and 12 after MRI examination. The International Cartilage Repair Society (ICRS) macroscopic score standard was used (Table S2) to assess the degree of defect regeneration. The harvested cartilages were fixed by 4% paraformaldehyde and then decalcified with 10% ethylenediaminetetraacetic acid for 3 months.
After embedding in paraffin, H&E, Safranin O, Toluidine blue, and Prussian blue staining of the samples sections (5 μm) were conducted to evaluate the histology.

| Statistical analyses
The data are expressed as the mean ± SD (n = 3). All statistical computations were exhibited by SPSS software (ver. 20.0; SPSS Inc., Chicago, IL). The homogeneity of variance was analyzed with F-test.
Differences in different groups were analyzed with one-way analysis of variance (ANOVA) with Bonferroni as post-hoc analyses for multiple comparisons. Repeated measurement ANOVA was used to evaluate differences among different time intervals for in vivo MRI experiments. Significant differences were presented as *P < 0.05, **P < 0.01, or ***P < 0.001.